1. Field of the Invention
This invention relates generally to biological assays, and more specifically to assay reagents labeled with fluorescent materials which reagents can be “toggled” from an intramolecular dimer to a fluorescent monomer by antibody binding.
2. State of the Art
Most clinical assays (e.g., immunoassays, DNA probe assays) are heterogeneous and consist of at least two steps: the binding of an antigen to its antibody, followed by physical separation of the bound from free antigens. In some more sensitive assays (e.g., “ELISA” or “EIA”) multiple steps are required. Homogeneous immunoassays, on the other hand, can distinguish between bound antigens and free ones without the need of additional separation steps. They are simple, rapid, yet more precise, more cost effective, and have the potential for total automation. For these reasons, separation-free assays are preferred in many applications such as biosensors, bioprobes and other automated instrumentation. J. P. Gosling, Clin. Chem., 36:1408–1427 (1990), D. W. Chan and M. T. Perlstein, Eds., Immunoassay, A Practical Guide (Academic Press, New York, 1987), and E. F. Ullman and P. L. Khanna, Methods in Enzymology, 74:28–60 (1981).
However, because of various technical complications homogeneous systems have been difficult to obtain, with the exception of a few assays suitable only for small molecules. J. F. Burd et al., Clin. Chem., 23:1402–1408 (1977), M. E. Jolley et al., Clin. Chem., 27:1190–1197 (1981), and D. L. Morris et al., Anal. Chem., 53:658–665 (1981).
It would be an improvement in the art to develop and characterize new fluorogenic tracer antigens that can be used as “reporter molecules” for the binding event without the need of separation steps and the labeling of antibodies. The development of such tracers could greatly facilitate the automation of a large array of clinical assays, especially of high molecular weight antigens. It would help reduce the operational time and cost, and make such assays more readily accessible to doctors and patients. Also, such tracers would be extremely useful for rapidly screening large numbers of recombinant antibodies generated with genetic engineering techniques, such as those described in C. F. Barbas et al., Proc. Natl. Acad. Sci. USA 89:4457–4461 (1992), R. A. Lemer et al., Science 258:1313–1314 (1992), and Marks et al. J. Biol. Chem. 267:16007–16010 (1992).